1. Field of the Invention
Hybrid DNA technology permits the transcription and translation of genes to produce desired polypeptide products. For the most part, the polypeptide products that are of interest are associated with mammalian genes, while the choice of host cells has been primarily directed to prokaryotes and lower eukaryotes, e.g. fungi, especially yeast due to ease of propagation and genetic manipulation. In developing a methodology for producing DNA constructs which provide efficient production of a desired polypeptide, one must develop a protocol which allows for the insertion and excision of DNA fragments in an ordered way. Frequently, the various regulatory signals, replication system, markers and the structural gene(s) of interest will come from diverse sources. Therefore, one must develop a strategy which allows for endonuclease cleavages, insertions and excisions in an appropriate sequence, which provides for the DNA fragments being aligned in the correct orientation and the proper spatial relationship. Furthermore, the presence of sequences which do or could interfere with the efficient transcription, translation or propagation of the desired structural gene, must be avoided or may be present only where they do not have a detrimental effect.
In addition to the strategy involved with the preparation of the DNA construct, there is also the considerations of the choice of sequences which encode for the regulatory signals and expression products, their individual interactions, their effect on the host, the efficiency with which the energy of the host is diverted to the production of the desired polypeptide, and the ease of isolation of the polypeptide product. Therefore, each development of a process for the production of a particular polypeptide becomes an intellectual exercise in experimental design, procedural organization, and investigation.
2. Description of the Prior Art
The human DNA sequence for human insulin may be found in Bell et al., Nature (1979) 282:525-527. Production of rat "pre"-proinsulin polypeptide fragments in bacteria is described in U.S. Pat. No. 4,338,397. Kurjan and Herskowitz, Cell (1982) 30:933-943 describe a putative .alpha.-factor precursor containing four tandem copies of mature .alpha.-factor, describing the sequence and postulating a processing mechanism. Kurjan and Herskowitz, Abstracts of Papers presented at the 1981 Cold Spring Harbor Meeting on The Molecular Biology of Yeast, p. 242, in an Abstract entitled, "A Putative Alpha-Factor Precursor Containing Four Tandem Repeats of Mature Alpha-Factor," describe the sequence encoding for the .alpha.-factor and spacers between two of such sequences. U.S. Pat. No. 4,338,397, col. 3 and 4, provides for useful definitions, which definitions are incorporated herein by reference.